TITLE: DETERMINATION OF THE KM, VMAX FOR AN ENZYMATIC REACTION:

THE REACTION OF TRYPSIN WITH BENZOYL-ARGININE-P-NITROANALIDE

NAME: Tadhg Barrett
STUDENT NUMBER: 20258887
MODULE: BC4904
COURSE: Industrial Biochemistry
 1. Introduction
Trypsin is a digestive enzyme which breaks down proteins in the small intestine, secreted
by the pancreas as trypsinogen. The substrate is BAPNA, a synthetic peptide, and pepsin
that usually hydrolyses polypeptides found in food. The presence of pepsin hydrolyses
polypeptides into peptides and free amino acids. Km is the substrate concentration at
which the reaction rate is 50% of the Vmax and Vmax is the maximum rate of the
enzyme catalysed reaction i.e. When the enzyme is saturated by the substrate. The Km
value for an enzyme depends on the particular substrate and on environmental conditions
such as pH, temperature and ionic strength. An enzyme with a high Km has a low affinity
meaning the enzyme will be slow acting, for its substrate and requires a greater
concentration of substrate to achieve Vmax. Kinetics is a method for determining how
quick and active a given enzyme is. The rate of change of product generated as a function
of time can be noticed in enzyme kinetics as a change in absorbance. By monitoring
absorbance change at 410nm in the case of trypsin and its substrate Benzoyl-arginine-p-
nitroanilide. The mechanism of action of a drug can be determined by monitoring enzyme
kinetics in the presence or absence of an inhibitor. Km and Vmax are calculated by
incubating the enzyme with various substrate concentrations and plotting the results as a
rate of reaction vs. substrate concentration graph. Experimental pharmacological
inhibitors of enzymes are tested using kinetics. If a medicine is effective, it should either
completely or partially change the kinetics of a response. The Km of the enzyme Trypsin
is calculated experimentally in this practical. The methods used to determine the kinetic
parameters should be documented so that a similar process can be devised to determine
these parameters for any enzyme. The hydrolysis of peptide bonds on the carboxyl side of
the basic amino acids L-arginine and L-lysine is catalysed by trypsin, an enzyme present
in pancreatic juice. The synthetic substrate Benzoyl-arginine-p-nitroanilide can be used to
readily monitor trypsin activity. This substance has a peptide-like structure. The enzyme
catalyses the release of p-nitroanilide from BAPNA, resulting in a rise in 410 nm
absorbance. Monitoring the absorbance as the digestion progresses can help you keep
track of what's going on.
2. Aim
Determine the Km and Vmax values of trypsin when it reacts with benzoyl-arginine-p-
nitroanilide

3. Materials and methods

Equipment

• Spectrophotometer Automatic pipettes Timer Cuvettes

Reagents
• Substrate Benzoyl-arginine-p-nitroanilide (Stock solution 200 mM)

• Trypsin (40 mg/mL).

• 0.1 M Tris-HCl -0. 02 M CaCl2 adjusted to pH 8.2

Protocol

1. Set up the spectrophotometer to measure at 410 nm and zero using a water blank.

2. Decide on the final concentrations of substrate required and calculate the volume of stock
solution to add.

3. Place in a 1 mL cuvette 0.8 mL Tris/CaCl2 buffer, 0.04 mL trypsin and distilled water such
that the final volume after addition of substrate is 1 mL.

4. Then add substrate, mix well by inversion, then replace the cuvette and measure and record
the change in absorbance at 410 nm at 30sec intervals.

5. Continue the measurement until the slope can be measured reasonably accurately (usually
less than 5 mins).

6. IMPORTANT: for each concentration of substrate set up an assay without enzyme to

determine the rate of spontaneous hydrolysis of substrate

4. Results
Table 1: dilution of substrate required

Sub a 1um = 0.8 Tris/CaCl2 buffer + 0.04 trypsin + 0.01 substrate = 0.85 – 1 = 0.15ml distilled
water
Sub b 2um = 0.8 Tris/CaCl2 buffer + 0.04 trypsin + 0.02 substrate = 0.86 – 1 = 0.14ml distilled
water
Sub c 4um = 0.8 Tris/CaCl2 buffer + 0.04 trypsin + 0.04 substrate = 0.88 – 1 =0.12ml distilled
water
Sub d 8um = 0.8 Tris/CaCl2 buffer + 0.04 trypsin + 0.08 substrate = 0.92 – 1 = 0.8ml distilled
water
Sub e 10um = 0.8 Tris/CaCl2 buffer + 0.04 trypsin + 0.1 substrate = 0.94 – 1 = 0.6ml distilled
water

Table 2: Absorbance values at 410 nm after 30s intervals:

Sub A:
 30-1.405
60-1.756
90-1.805
120-2.244
150-2.564
180-2.803
210-2.941
240-3.066

sub A
3.5

3 f(x) = 0.00828015873015873 x + 1.20517857142857

R² = 0.973837260694438
Absorbance @410nm

2.5

1.5

0.5

0
0 50 100 150 200 250 300
time (seconds)

Sub B:

30-1.906
60-2.315
90-2.519
120-2.744
150-.984
180-3.289
210-3.356
240-3.453
 Sub B
4
3.5
f(x) = 0.00737460317460317 x + 1.82517857142857
3 R² = 0.968943242584497
Absorbance@410nm

2.5
2
1.5
1
0.5
0
0 50 100 150 200 250 300
time(seconds)

Sub C:

30-2.345
60-2.655
90-2.891
120-3.089
150-3.245
180-3.455
210-3.541
240-3.688

Sub C
4
3.5 f(x) = 0.0062218253968254 x + 2.27367857142857
R² = 0.977056017449019
3
Absorbance@410nm

2.5
2
1.5
1
0.5
0
0 50 100 150 200 250 300
time(seconds)

Sub D:

30-2.545
60-2.741
 90-2.963
120-3.162
150-3.357
180-3.565
210-3.741
240-3.963

Sub D
4.5
4
f(x) = 0.00671706349206349 x + 2.34782142857143
3.5 R² = 0.999603868134552
Absorbance@410nm

3
2.5
2
1.5
1
0.5
0
0 50 100 150 200 250 300
time(seconds)

Sub E:

30-2.741
60-2.954
90-3.046
120-3.364
150-3.499
180-3.674
210-3.841
240-4.041
 Sub E
4.5
4
f(x) = 0.00617222222222222 x + 2.56175
3.5 R² = 0.99323595760843
Absorbance @410nm

3
2.5
2
1.5
1
0.5
0
0 50 100 150 200 250 300
time(seconds)

5. Discussion
The relationship at 410nm of the product and the concentration of the trypsin in the
sample is directly proportional, therefore a straight-lined graph through the origin
should be expected once the date was graphed. However, my results didn’t show this.
The influence of substrate concentration on enzyme activity is discussed in the first
portion of this lab report. Each graph depicts the absorbance level of each substrate
over a 4-minute period, split down into 30-second intervals. We can see from the
graphs that as our solution's substate concentration increased, so did the absorbance
levels. The graphs show that when the concentration of substrate increases, the rate of
reaction increases. This is due to the increased likelihood of a substrate and enzyme
colliding. The reason for this is that there are far more substrates than enzymes, and
all enzymes are stated as being fully saturated. At this point, the process has reached
its maximum speed and cannot be sped up without the addition of more enzymes,
hence enzyme concentration is the limiting factor.
6. Conclusion
In conclusion, a series of graphs were constructed by measuring the absorbance at
410nm of BAPNA produced in the presence of different known concentrations of
trypsin.